CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-¹⁴C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.     

Comparison of reagent-impregnated paper strips and conventional tests for distinguishing Escherichia from Aerobacter: correlation with colonial morphology

The means for distinguishing Escherichia from Aerobacter (Enterobacter) differ in laboratories and range from complete dependence on colonial reactions on typical gram-negative media to reliance on one or more of the classical indole, methyl red, Voges-Proskauer, citrate (IMViC) parameters. Three colonial types (one prejudged as Escherichia) of lactose-positive rods were catalogued on each of the most commonly used selective media, MacConkey Agar, Endo Agar, and E M B Agar. Each cultural type was presumptively diagnosed and then compared with the expected outcome of individual IMViC tests. The distribution of preliminary identifications was similar from growth patterns on MacConkey Agar and E M B Agar, but it differed markedly from Endo Agar. When organisms initially diagnosed by cultural methods were compared by single IMViC tests, it was found that for each colonial type one of the biochemical parameters was best suited. Thus, for those types initially considered Escherichia, the methyl red or Voges-Proskauer test results agreed most consistently; for other types, the citrate reaction was most satisfactory. In addition, when newly formulated reagent-impregnated paper strip methods for indole, Voges-Proskauer, and citrate were evaluated and compared to the standard methods, agreement was 97% for indole, 90% for Voges-Proskauer, and 95% for Simmons' citrate.     

New Diagnostic System for the Identification of Lactose-fermenting Gram-negative Rods

The identification of prompt lactose-fermenting gram-negative rods has generally relied heavily upon colonial morphology coupled with one or more indole, methyl red, Voges-Proskauer, citrate (IMViC) parameters, hydrogen sulfide, and motility. Studies were undertaken to compare diagnoses dependent solely upon the more orthodox criteria to a system for identification based upon hydrogen sulfide, ornithine decarboxylase, and citrate utilization (HOC). The results suggest that the IMViC scheme of identification is neither consistent nor applicable when applied to the current nomenclature of the above group of organisms and should be discarded, whereas the HOC system may prove to be of significant value to clinical microbiologists.     

Depolarization-induced release of [3H]histamine by high potassium concentrations,electrical stimulation and veratrine from rat brain slices after incubation with the radiolabelled amine

Brain slices obtained from neocortex, hypothalamus or hippocampus were incubated with [³H]histamine and subsequently superfused and exposed to different depolarizing stimuli, viz. high K⁺-concentrations, electrical field stimulation and veratrine. K⁺-induced release of tritium was completely calcium-dependent and its magnitude depended on the K⁺-concentration, with maximal release being reached at 56 mM K⁺. Electrically-evoked release of tritium increased with increasing frequencies and reached its maximum at about 20 Hz. The electrically-evoked release appeared to be totally calcium-dependent and it was strongly inhibited by tetrodotoxin. Veratrine (5–100 μM) also induced a release of tritium; maximal release was obtained at 100 μM veratrine. Veratrine-induced release was partially calcium-dependent and was strongly reduced by tetrodotoxin.Taken together the data indicate that the depolarization-induced release of tritium from brain slices pre-labelled with [³H]histamine, represents [³H]histamine release from neurons and not from either mast cells or glial cells. It remains to be established whether these neurons are specifically histaminergic.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues

Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I₅₀ = 2 μg/ml) when compared to other naturally occurring glycosaminoglycans. This inhinibition was also appparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E ₂. Heparin was also found to inhibit glucagon-sensitive rat hepatice adenylate cyclase, and the prostaglandin E₁-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfade polysugar dextran sulfate exerts similar effects on adenylate cyclase activity of the rat ovary was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.     

Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms

A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.     

A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney

Polycystic kidney disease (PKD) is a common human genetic illness. It is characterized by the formation of multiple kidney cysts that are thought to result from over-proliferation of epithelial cells. Zebrafish larvae can also develop kidney cysts. In an insertional mutagenesis screen in zebrafish, we identified 12 genes that can cause cysts in the glomerular-tubular region when mutated and we cloned 10 of these genes. Two of these genes, vhnf1 (tcf2) and pkd2, are already associated with human cystic kidney diseases. Recently, defects in primary cilia have been linked to PKD. Strikingly, three out of the 10 genes cloned in this screen are homologues of Chlamydomonas genes that encode components of intraflagellar transport (IFT) particles involved in cilia formation. Mutation in a fourth blocks ciliary assembly by an unknown mechanism. These results provide compelling support for the connection between cilia and cystogenesis. Our results also suggest that lesions in genes involved in cilia formation and function are the predominant cause of cystic kidney disease, and that the genes identified here are excellent candidates for novel human PKD genes.     

Dual 5-HT1A agonists and 5-HT re-uptake inhibitors by combination of indole-butyl-amine and chromenonyl-piperazine structural elements in a single molecular entity

The dual serotonin (5-HT) re-uptake inhibitor and 5-HT(1A) receptor agonist vilazodone was found to increase central serotonin levels in rat brain. In the course of structural modifications of vilazodone 3-[4-[4-(2-oxo-2H-1-benzopyran-6-yl)-1-piperazinyl]-butyl]-1H-indole-5-carbonitrile 8i and its fluorine analogue 6-[4-[4-(5-fluor-3-indolyl)-butyl]-1-piperazinyl]-2H-1-benzopyran-2-one have been identified. These unsubstituted chromenones are equally potent at the 5-HT(1A) receptor and 5-HT transporter. The implementation of nitrogen functionalities in position 3 of the chromenones resulted in compounds acting as agonists at the 5-HT(1A) receptor and as 5-HT re-uptake inhibitors like vilazodone. Ex vivo 5-HT re-uptake inhibition and in vitro 5-HT agonism were determined in the PCA- and GTPgammaS-assay, respectively. The potential of these chromenones to increase central 5-HT levels was measured in microdialysis studies and especially the derivatives 3-[4-[4-(3-amino-2-oxo-2H-chromen-6-yl)-piperazin-1-yl]-butyl]-1H-indole-5-carbonitrile 8f, ethyl (6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-carbamate 8h and N-(6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-acetamide 8k give rise to rapid development of increased serotonin levels in rat brain cortex, lasting longer than 3h.